Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids

Background: Individuals carrying the factor V Leiden mutation have been sho wn to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic a cids (LNAs). Methods: LNA octamer probes complementary to the factor V wild-type or muta ted sequence were covalently attached to individual wells of a microtiter p late. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish pero xidase-antibiotin Fab conjugate and tetramethylbenzidine substrate. Results: In a prospective study of 53 patients, the assay reproducibly scor ed both factor V homozygotes and heterozygotes with excellent sensitivity a nd specificity. All results were in complete agreement with the results obt ained with the conventional PCR-restriction fragment length polymorphism te chnique. Conclusions: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V tes ting in the clinical laboratory.